Burns often lead to persistent pain not well managed by available analgesics. The mechanisms of burn pain are incompletely understood. However, the TRPV1 (transient receptor potential V1) receptor appears to be pivotally involved in post-burn pain. In our preliminary data, ~67% of post-burn thermal hyperalgesia is blocked by peripheral injection of a TRPV1 receptor antagonist. In addition, extracts of burned human skin produce thermal hyperalgesia when injected into rats via a TRPV1 receptor mechanism. These novel data suggest that TRPV1 significantly contributes to post-thermal burn injuries. Interestingly, the precise mechanism(s) for the activation of TRPV1 remains unknown. Converging evidence indicates that oxidized lipids derived from linoleic or arachidonic acid are released during tissue injury and activate TRPV1 and/or TRPA1, resulting in nociceptor depolarization and pain. Many of these lipids are formed by the actions of enzymes such as cytochrome P450s (CYP) or lipoxygenase (LOX). This is of particular significance since analysis of the GLUE Grant indicates that transcripts encoding these enzymes are elevated as early as 0-3 days after burns. Therefore, we propose the central hypothesis that oxidized lipids are enzymatically formed after human burn injuries and contribute to post-burn pain by activation of TRPV1 and/or TRPA1. The present Aims will: Specific Aim 1: Identify enzymes that oxidize linoleic acid to form metabolites that activate TRPV1 or TRPA1. We have analyzed the NIGMS-supported GLUE Grant microarray database of transcripts from skin with burn injury vs. control human skin and found significantly elevated levels of CYPs and LOXs that persist for up to 12 months. This Aim will use a COS expression system to evaluate whether these enzymes are capable of oxidizing linoleic acid into TRPV1 or TRPA1 agonists using patch clamp electrophysiology and determine the functional activity of these compounds in preclinical studies. Additional studies will evaluate possible entourage interactions among these lipids for activating TRP channels. Specific Aim 2: Identify enzymes that oxidize arachidonic acid to form metabolites that activate TRPV1 or TRPA1. Aim 2 will use the same approach as Aim 1, but will focus on arachidonic acid metabolites. This novel hypothesis has strong scientific and medical implications. From a scientific perspective, it expands the focus of TRPV1 from being a detector of transient heat, to a detector of oxidized lipids released from burned skin long after the heat-induced injury. From a medical perspective, this hypothesis is innovative since it leads to a fundamentally new pharmacological approach for treating burn pain - by blocking the enzymatic synthesis of TRPV1- and/or TRPA1-active compounds released from tissues after burns. The present proposal is designed to comprehensively test this potentially ground-breaking hypothesis of persistent post-burn pain.